Ku70 Binding Protein-5 (KUB5), a Novel Factor in Nonhomologous End Joining

Ku70 结合蛋白-5 (KUB5)，非同源末端连接的新因子

• 批准号: 8100383
• 负责人: David A Boothman
• 金额: $ 31.9万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8100383
• 关键词: 5' Untranslated Regions; ATM activation; Affect; Binding; Binding Proteins; Biochemical; Breast; Breast Cancer Cell; C-terminal; Cell Line; Cell physiology; Cells; Chromatids; Chromatin Loop; Cisplatin; Coiled-Coil Domain; Comet Assay; Complementary DNA; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA repair protein; DNA-Directed RNA Polymerase; Data; Defect; Depressed mood; Development; Diploidy; Double Strand Break Repair; Drug resistance; Embryo; Exonuclease; Failure; Fatty acid glycerol esters; Fibroblasts; Future; G22P1 gene; Gel Chromatography; Genes; Genetic; Genetic Transcription; Genomic Instability; Genomics; Growth; Haploidy; Heterozygote; Homologous Gene; Human; Hybrids; Hypersensitivity; Ionizing radiation; Knock-out; Knockout Mice; Ligation; Link; Malignant Neoplasms; Malignant neoplasm of ovary; Mammalian Cell; Mediating; Modeling; Monitor; Mus; N-terminal; Nature; Nonhomologous DNA End Joining; Nuclear Receptor Coactivator 3; Phase; Phenotype; Play; Process; Proteins; RNA; RNA Polymerase II; Radio; Regulation; Reporting; Resistance; Ribonuclease H; Role; Serine; Site; Small Interfering RNA; Source; Structure; Testing; Transcript; Untranslated Regions; Western Blotting; Yeasts; artemis; cancer cell; chemotherapy; homologous recombination; in vivo; killings; malignant breast neoplasm; mutant; novel; prevent; protein function; public health relevance; radiation resistance; repaired; resistance mechanism; response; small hairpin RNA; transcription termination; vector; yeast two hybrid system

DESCRIPTION (provided by applicant): Alterations in DNA repair play major roles in breast cancer resistance to ionizing radiation (IR) and chemotherapies. Factors operating in both DSB repair and RNA transcription, specifically proteins regulating RNA polymerase (Pol) II, have been suggested, but few reported. When RNA Pol II stalls, or when transcription cannot properly terminate, RNA/DNA hybrids (R-loops) have extended half-lives that result in DNA double strand break (DSB) formation and genetic instability. Using yeast two hybrid analyses, we identified Ku70 binding protein #5 [(Kub5)/Hera], a highly conserved human homolog of yeast rtt103 that terminates RNA transcription via its regulation of RNA Pol II. Stable loss of Kub5/Hera expression, either by heterozygote knockout or knockdown by siRNA/shRNA expression, leads to elevated basal DSBs, ATM activation, foci formation and chromatid aberrations that were abrogated by RNase H forced over- expression, suggesting a role for R-loops in genetic instability. Kub5/Hera knockdown cells were hypersensitive to IR, equivalent to Ku70 deficient cells, and failed to repair DSBs that require DNA end-processing. We hypothesize that KUB5/HERA plays dual roles in the cell to: (i) regulate RNA Pol II to terminate RNA transcription via its interaction with the RNA Pol II CTD domain; and (ii) stimulate NHEJ processing of complex DSBs by Ku70 and Artemis interactions. When its expression is depressed (e.g., one-half via haplo-insufficiency) R-loops form and DSBs are created due to deficient RNA transcription termination. DNA damage is simultaneously amplified by the cell's inability to repair DSBs downstream, leading to IR hypersensitivity. Three Specific Aims are proposed: Aim 1: to perform structure/function analyses of Kub5/Hera focusing on two regions, the RPR and coiled-coil domains, to uncouple transcription termination and DSB repair; Aim 2: to define functions of KUB5- Ku70-Artemis complexes in DSB repair; and Aim 3: to define the role(s) of KUB5/HERA in regulating RNA Pol II function, RNA transcription termination, and spontaneous DSB formation and chromatid aberrations. These studies will allow us to explore the role of KUB5/HERA over-expression in human breast cancer radio-resistance. PUBLIC HEALTH RELEVANCE: Human Ku70 binding protein #5, also known as HERA, (KUB5/HERA) is a new factor involved in the repair of DNA ends with complex damage, such as blunt-end breaks. Elevations of KUB5/HERA in breast and ovarian cancers, and our demonstration that KUB5/HERA is extremely important for drug and radiation resistance, suggests that this protein is a valuable target for future therapies and a 'valuable predictor of response' for therapies. Our recent observation that KUB5/HERA is required for RNA termination allows us to also investigate the interface between DNA repair and regulation of RNA Polymerase II and transcription termination.

描述（由申请人提供）：DNA 修复的改变在乳腺癌对电离辐射 (IR) 和化疗的抵抗中发挥着重要作用。已经提出了在 DSB 修复和 RNA 转录中起作用的因素，特别是调节 RNA 聚合酶 (Pol) II 的蛋白质，但很少有报道。当 RNA Pol II 停滞或转录无法正常终止时，RNA/DNA 杂合体（R 环）的半衰期延长，导致 DNA 双链断裂 (DSB) 形成和遗传不稳定。 使用酵母两种杂交分析，我们鉴定了 Ku70 结合蛋白 #5 [(Kub5)/Hera]，这是酵母 rtt103 的高度保守的人类同源物，可通过 RNA Pol II 的调节终止 RNA 转录。通过杂合子敲除或 siRNA/shRNA 表达敲低，Kub5/Hera 表达的稳定丧失会导致基础 DSB 升高、ATM 激活、病灶形成和染色单体畸变，而这些都被 RNase H 强制过度表达所消除，这表明 R 环在遗传不稳定中发挥作用。 Kub5/Hera 敲低细胞对 IR 高度敏感，相当于 Ku70 缺陷细胞，并且无法修复需要 DNA 末端处理的 DSB。 我们假设 KUB5/HERA 在细胞中发挥双重作用：（i）通过与 RNA Pol II CTD 结构域的相互作用来调节 RNA Pol II 以终止 RNA 转录； (ii) 通过 Ku70 和 Artemis 相互作用刺激 NHEJ 对复杂 DSB 的处理。当其表达受到抑制（例如，由于单倍体不足而降低一半）时，由于 RNA 转录终止缺陷，就会形成 R 环并产生 DSB。由于细胞无法修复下游 DSB，DNA 损伤同时被放大，导致 IR 超敏反应。提出了三个具体目标： 目标 1：对 Kub5/Hera 进行结构/功能分析，重点关注 RPR 和卷曲螺旋结构域这两个区域，以解开转录终止和 DSB 修复；目标2：明确KUB5-Ku70-Artemis复合物在DSB修复中的功能；目标 3：确定 KUB5/HERA 在调节 RNA Pol II 功能、RNA 转录终止、自发 DSB 形成和染色单体畸变中的作用。这些研究将使我们能够探索 KUB5/HERA 过度表达在人类乳腺癌放射抗性中的作用。 公共健康相关性：人类 Ku70 结合蛋白 #5，也称为 HERA，(KUB5/HERA) 是一种新因子，参与修复具有复杂损伤（例如平末端断裂）的 DNA 末端。 乳腺癌和卵巢癌中 KUB5/HERA 的升高，以及我们证明 KUB5/HERA 对于药物和放射耐受性极其重要，表明该蛋白是未来治疗的有价值的靶标，也是治疗的“有价值的反应预测因子”。我们最近观察到 KUB5/HERA 是 RNA 终止所必需的，这使我们能够研究 DNA 修复和 RNA 聚合酶 II 的调节以及转录终止之间的界面。